Nucleic acids, proteins and polysaccharides constitute the three major classes of biopolymers. While the first two systems are principally linear assemblies, polysaccharides are structurally more complex. This structural and stereochemical diversity results in a rich content of “information” in relatively small molecules. Nature further “leverages” the structural biomolecules such as isoprenoids, fatty acids, neutral lipids, peptides or proteins. Oligosaccharides in the form of glycoconjugates mediate a variety of events including inflammation, immunological response, metastasis and fertilization. Cell surface carbohydrates act as biological markers for various tumors and as binding sites for other substances including pathogens.
Proteoglycans are complex protein-carbohydrate assemblies that consist of a core protein and one or more covalently attached glycosaminoglycan chains. For reviews, see: R. V. Iozzo, Annu. Rev. Biochem. 1998, 67, 609-652; and M. Bemfield, M. Gotte, P. W. Park, O. Reizes, M. L. Fitzgerald, J. Lincecum, M. Zako, Annu. Rev. Biochem. 1999, 68, 729-777. These linear polysaccharides range in length from ˜20 to 200 disaccharide repeat units, each composed of an amino sugar and an uronic acid moiety (FIG. 1).
Heparin-like glycosaminoglycans (HLGAGs) are the most acidic naturally occurring biopolymers. These complex polysaccharides, found in the extracellular matrix, play a key role in regulating the biological activity of several proteins in the coagulation cascade along with many other processes of biomedical importance including growth factor interactions, virus entry, and angiogenesis. H. E. Conrad, Heparin Binding Proteins; Academic Press 1998. Heparin, isolated from the mast cells of pigs, is currently produced in multi-ton quantities and used in a variety of medical applications. H. Engelberg, Pharmacol. Rev. 1984, 36, 91-110. Most prominent is the use of heparin as an anticoagulant in heart disease where it has served as a therapeutic agent since the late 1930s. The heterogeneity of heparin results in many severe side effects, making this inexpensive drug dangerous and necessitates close monitoring. B. H. Chong, Aust. N.Z. J Med. 1992, 22, 145-152.
The heparin-antithrombin III (AT-III) interaction is responsible for heparin's anticoagulant activity and is the only system where the exact sequence of heparin that associates with the protein has been identified. Extensive structure-activity studies using synthetic oligosaccharides (M. Petitou, P. Duchaussoy, G. Jaurand, F. Gourvenec, I. Lederman, J.-M. Strassel, T. Barzu, B. Crepon, J.-P. Herault, J.-C. Lormeau, A. Bernat, J.-M. Herbert, J. Med. Chem. 1997, 40, 1600-1607; and S. Koshida, Y. Suda, M. Sobel, J. Ormsby, S. Kusumoto, Bioorg. Med. Chem. Lett. 1999, 9, 3127-3132.) as well as NMR (M. Iacomini, B. Casu, M. Guerrini, A. Naggi, A. Pirola, G. Torri, Anal. Biochem. 1999, 274, 50-58.) and X-ray crystallography (S. Faram, R. E. Hileman, J. R. Fromm, R. J. Linhardt, D. C. Rees, Science 1996, 271, 1116-1120.) have been performed. Based on these studies, a concerted drug development effort has been undertaken, resulting in the development of a synthetic pentasaccharide heparin analog for use in humans. M. Petitou, P. Duchaussoy, P. A. Driguez, G. Jaurand, J. P. Herault, J. C. Lormeau, C. A. A. van Boeckel, J. M. Herbert, Angew. Chem. Int. Ed. 1998, 37, 3009-3014, Angew. Chem. 1998, 110, 3186-3191; and B. Mulloy, M. J. Forster, Glycobiology 2000, 10, 1147-1156. With the exception of the AT-III-heparin interaction, the relationship between structure and function of HLGAGs is still poorly understood due to the complexity and heterogeneity of these polymers. Defined HLGAG oligosaccharides constitute valuable molecular tools to gain a detailed understanding of the sequences of HLGAGs responsible for binding to a particular protein and modulating its biological activity. For reviews, see: R. V. Iozzo, Annu. Rev. Biochem. 1998, 67, 609-652; and M. Bernfield, M. Gotte, P. W. Park, O. Reizes, M. L. Fitzgerald, J. Lincecum, M. Zako, Annu. Rev. Biochem. 1999, 68, 729-777. Determination of the structure-activity relationships of HLGAGs will create an opportunity for the discovery of novel therapeutic interventions for many disease states.
Over the past two decades, a variety of synthetic methods directed at the preparation of HLGAG oligosaccharides have been disclosed and heroic total syntheses (P. Sinay, J.-C. Jacquinet, Carbohydr. Res. 1984, 132, C5-C9; M. Petitou, P. Duchaussoy, I. Lederman, J. Choay, P. Sinay, J.-C. Jacquinet, D. Iorri, Carbohydr. Res. 1986, 147, 221-236.) have resulted in the assembly of AT-III-binding HLGAG oligosaccharides. C. A. A. van Boeckel, M. Petitou, Angew. Chem. Int. Ed. Engl. 1993, 32, 1671-1690, Angew. Chem. 1993, 105, 1741-1761; P. Westerduin, J. E. M. Basten, M. A. Broekhoven, V. de Kimpe, W. H. A. Kuijpers, C. A. A. van Boeckel, Angew. Chem. Int. Ed. Engl. 1996, 35, 331-333, Angew. Chem. 1996, 108, 339-342. More recently, longer oligosaccharide HLGAG analogs exhibiting impressive biological activity have been prepared using simplified syntheses. M. Petitou, J.-P. Herault, A. Bernat, P.-A. Driguez, P. Duchaussoy, J.-C. Lormeau, J.-M. Herbert, Nature, 1999, 398, 417-422. Still, the procurement of specific HLGAG sequences required the development of a new total synthesis stratgey for each oligosaccharide target.
Moreover, many additional physiologically-important recognition phenomena involving carbohydrates have been discovered in recent years. Lectins, proteins which contain carbohydrate recognition domains, have been identified. Prominent members of the calcium dependent (C-type) lectin family (Drickamer, K. Curr. Opin. Struct. Biol. 1993, 3, 393) are the selectins which play a crucial role in leukocyte recruitment in inflammation. Bevilacqua, M. P.; Nelson, R. M. J. Clin. Invest. 1993, 91, 379. Members of the C-type lectin superfamily have been described on NK cells and Ly-49, NKR-P1 and NKG2 constitute group V of C-type lectins. While many lectins have been purified and cloned, their ligands have not been identified due to the heterogeneous nature of carbohydrates.
The recognition that interactions between proteins and carbohydrates are involved in a wide array of biological recognition events, including fertilization, molecular targeting, intercellular recognition, and viral, bacterial and fungal pathogenesis, underscores the importance of carbohyrates in biological systems. It is now widely appreciated that the oligosaccharide portions of glycoproteins and glycolipids mediate certain recognition events between cells, between cells and ligands, between cells and the extracellular matrix, and between cells and pathogens. See, e.g., U.S. Pat. No. 4,916,219 (describing oligosaccharides with heparin-like anticomplement activity).
These recognition phenomena may be inhibited by oligosaccharides having the same sugar sequence and stereochemistry found on the active portion of a glycoprotein or glycolipid involved in the recognition phenomena. The oligosaccharides are believed to compete with the glycoproteins and glycolipids for binding sites on the relevant receptor(s). For example, the disaccharide galactosyl-β-1-4-N-acetylglucosamine is believed to be one component of the glycoproteins which interact with receptors in the plasma membrane of liver cells. Thus, to the extent that they compete with moieties for cellular binding sites, oligosaccharides and other saccharide compositions have the potential to open new horizons in pharmacology, diagnosis, and therapeutics.
The growing appreciation of the key roles of oligosaccharides and glycoconjugates in fundamental life sustaining processes has stimulated a need for access to usable quantities of these materials. Glycoconjugates are difficult to isolate in homogeneous form from living cells since they exist as microheterogeneous mixtures. The purification of these compounds, when possible, is at best tedious and generally provides only very small amounts of the compounds. The travails associated with isolation of oligo- and poly-saccharides and glycoconjugates from natural sources present a major motivation for the development and exploitation of chemical synthesis. See, e.g., U.S. Pat. Nos. 4,656,133; 5,308,460; 5,514,784; and 5,854,391 (describing various means of glycosylating saccharides and peptides).
Intense work on the further development of the use of biologically-active oligosaccharides is ongoing within a number of fields, including: novel diagnostics and blood typing reagents; highly specific materials for affinity chromatography; cell specific agglutination reagents; targeting of drugs; monoclonal antibodies, e.g., against cancer-associated reagents; antibiotic alternatives, based on the inhibition with specific oligosaccharides of the attachment of bacteria and viruses to cell surfaces; and stimulation of the growth of plants and protection of them against pathogens.
The invention of solid phase peptide synthesis by Merrifield 35 years ago dramatically influenced the strategy for the synthesis of biopolymers. The preparation of structurally defined oligopeptides (Atherton, E.; Sheppard, R. C. Solid phase peptide synthesis: A practical approach; IRL Press at Oxford University Press: Oxford, England, 1989, pp 203) and oligonucleotides (Caruthers, M. H. Science 1985, 230, 281) has benefited greatly from the feasibility of conducting their assembly on various polymer supports. The advantages of solid matrix-based synthesis, in terms of allowing for an excess of reagents to be used and in the facilitation of purification are now well appreciated. However, the level of complexity associated with the synthesis of an oligosaccharide on a polymer support dwarfs that associated with the other two classes of repeating biooligomers. First, the need to differentiate similar functional groups (hydroxyl and amino) in oligosaccharide construction is much greater than the corresponding needs in the synthesis of oligopeptides or oligonucleotides. Furthermore, in these latter two cases, there is no stereoselection associated with construction of the repeating amide or phosphate bonds. In contrast, each glycosidic bond fashioned in a growing oligosaccharide ensemble constitutes a new locus of stereogenicity.
Combinatorial chemistry has been used in the synthesis of large numbers of structurally distinct molecules in a time and resource-efficient manner. Peptide, oligonucleotide, and small molecule libraries have been prepared and screened against receptors or enzymes to identify high-affinity ligands or potent inhibitors. These combinatorial libraries have provided large numbers of compounds to be screened against many targets for biological activity. Every pharmaceutical company now devotes a major effort to the area of combinatorial chemistry in order to develop new lead compounds in a rapid fashion.
The development of protocols for the solid support synthesis of oligosaccharides and glycopeptides requires solutions to several problems. Of course, considerable thought must be addressed to the nature of the support material. The availability of methods for attachment of the carbohydrate from either its “reducing” or “non-reducing” end would be advantageous. Also, selection of a linker which is stable during the synthesis, but can be cleaved easily when appropriate, is critical. A protecting group strategy that allows for high flexibility is desirable. Most important is the matter of stereospecific and high yielding coupling reactions.
Combinatorial carbohydrate libraries also hold a tremendous potential with regard to therapeutic applications. The key role complex oligosaccharides play in biological processes, such as inflammation, immune response, cancer and fertilization makes them highly attractive therapeutic targets. The ability to create true oligosaccharide libraries has the potential to trigger a revolution in the area of biopharmaceuticals. For example, the generation of combinatorial carbohydrate libraries will facilitate the rapid identification of ligands to many carbohydrate binding proteins which are involved in a variety of important biological events including inflammation (Giannis, A. Angew. Chem. Int. Ed. Eigl. 1994, 33, 178), immune response (Ryan, C. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1) and metastasis (Feizi, T. Curr. Opin. Struct. Biol. 1993, 3, 701). Analogs of ligands can help to define important lectin-ligand interactions. Non-natural ligands can be powerful inhibitors of carbohydrate-protein binding and will facilitate the study of cascade-like events involving such interactions. Furthermore, inhibitors of carbohydrate-lectin binding are potential candidates for a variety of therapeutic applications.
As stated above, due to the difficulties associated with purification of glycoconjugates and oligosaccharides from natural sources, chemical synthesis may be the only way to procure sufficient amounts of these structures for detailed biochemical and biophysical studies. Additionally, combinatorial carbohydrate libraries hold great potential for the identification of carbohydrate-based ligands to cellular receptors. Identification of these molecules will open many new avenues for the development of diagnostic tools and therapeutic agents.